Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide

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Effects of lipid composition on the entry of cellpenetrating peptide oligoarginine into single vesicles Sabrina Sharmin, Md. Zahidul Islam, Mohammad Abu Sayem Karal, Shibly Sayed Ul Alam, Hideo Dohra, and Masahito Yamazaki Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00189 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 17, 2016

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Biochemistry

Effects of lipid composition on the entry of cell-penetrating peptide oligoarginine into single vesicles Sabrina Sharmin,a Md. Zahidul Islam,a Mohammad Abu Sayem Karal,a,# Sayed Ul Alam Shibly,a Hideo Dohra,b and Masahito Yamazakia, c, d,*

a

Integrated Bioscience Section, Graduate School of Science and Technology, b Instrumental Research Support

Office, Research Institute of Green Science and Technology, c Nanomaterials Research Division, Research Institute of Electronics, d Department of Physics, Graduate School of Science, Shizuoka University, Shizuoka 422-8529, Japan #

Present address: Department of Physics, Bangladesh University of Engineering and Technology, Dhaka-1000,

Bangladesh

Funding This work was supported in part by a Grant-in-Aid for Scientific Research (B) (No.15H04361) from the Japan Society for the Promotion of Science (JSPS) to M.Y.

*Correspondence should be addressed to: Dr. Masahito Yamazaki Nanomaterials Research Division, Research Institute of Electronics, Shizuoka University 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan Tel/Fax: 81-54-238-4741 E-mail: [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with GUVs of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) containing AF647 and small vesicles composed of DOPG/DOPC. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was small. We obtained similar results for GUVs of dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8). The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8)-GUVs were larger than those obtained with DOPG/DOPC (2/8)-GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6)-GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was larger. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4)-GUVs. Analysis of the rim intensity showed that CF-R9 located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4)-GUVs. Based on analyses of these results, we discuss the elementary processes by which CF-R9 enters GUVs of various lipid compositions.


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Cell-penetrating peptides (CPPs) can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargos such as proteins and oligonucleotides.1-5 CPPs can be classified into two groups, amphipathic and non-amphipathic CPPs, based on their physicochemical characteristics. Penetratin, vascular endothelial cadherin protein, transportan (TP), and TP10 (a truncated analogue of TP), are amphipathic CPPs. Human immunodeficiency virus-1 Tat protein-derived peptide (positions 48-60) (here abbreviated Tat (48-60)), and oligoarginine containing many Arg residues, are non-amphipathic CPPs.6-9 The mechanism by which CPPs and their cargos translocate across the plasma membrane is still controversial. Some CPPs internalize via endocytosis, but others use non-endocytosis pathways.1-5 In both cases, CPPs must translocate across the lipid bilayer to enter a cell. It was recently reported that there is low efficiency of transfer of CPPs from endosomes to the cytosol if CPPs enter cells via endocytosis.10,11 Oligoarginine, abbreviated Rn or (Arg)n (n = 4−16), has been developed as a model peptide of Tat (48-60) and has been demonstrated to have cell-penetrating activity.9,12 Experiments using living (unfixed) cells demonstrated that R9 internalizes via endocytosis,8,13 although under some conditions, Rn can enter cells via a non-endocytosis pathway.14 To elucidate the mechanism by which Rn penetrates through plasma membranes, a number of researchers have investigated the interactions of Rn with lipid membranes.14-17 R9 binds to negatively charged membranes due to electrostatic interactions.15 It is also reported that hydrogen bonding between the guanidinium of Arg and the phosphate group of phospholipids plays an important role in the binding of Rn to phospholipid membranes.14,16 R9 in lipid membranes adopts a random-coil structure.17 The interactions of other CPPs such as TP10 with lipid membranes have been investigated using suspensions of large unilamellar vesicles (LUVs) of lipid membranes (the LUV suspension method).18-20 TP10 was shown to induce substantial leakage (diffusion) of a small water-soluble fluorescent probe, carboxyfluorescein, from the inside to the outside of LUVs in a concentration-dependent manner.18 However, the interaction of R9 for 30 minutes with LUVs that mimic the late endosomal membrane induced only 1% leakage of a small water-soluble fluorescent probe, calcein.21 Recently, Mishra et al. demonstrated that R6 induced rupture of giant unilamellar vesicles (GUVs) and leakage of fluorescent probes, and that fluorescein-labeled R6 (R6-FITC) induced substantial leakage of these fluorescent probes, suggesting that first R6-FITC formed pores in the membrane, then it entered the GUVs.22 Mishra et al. used lipid membranes

containing

high

concentrations

of

dioleoylphosphatidylethanolamine


(DOPE)

and

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dioleoylphosphatidylserine (DOPS), and the authors demonstrated that the interaction of Rn with DOPS/DOPE (2/8) membranes induces the inverse bicontinuous double-diamond cubic phase (QIID).22 It is well known that in the QIID phase, an infinite periodic minimal surface (IPMS), in which the surface has zero mean curvature and negative Gaussian curvature at all points, lies at the bilayer mid-plane.23,24 Therefore, the authors concluded that negative Gaussian curvature (i.e., saddle-splay) plays an important role in the entry of Rn into GUVs22 and also the entry of TAT (47-57) into GUVs.25 The formation of the cubic phase observed by Mishra et al. is associated with the particular lipid compositions (namely, high concentrations of DOPE) of the membranes they used in their investigation. It is generally believed that cubic phases are not induced in most lipid membranes upon interaction with Rn. We developed the single GUV method to investigate peptide/protein-induced pore formation in lipid membranes.26-30 In this method, changes in the structure and physical properties of a single GUV that are induced by its interactions with peptides/proteins are observed as a function of temporal and spatial coordinates. The same experiments are carried out using many “single GUVs” and the results are statistically analyzed. Thus, the single GUV method can reveal the details of elementary processes of individual events, and allow calculation of their kinetic constants. Using this method, we obtained information on the elementary processes of pore formation (e.g., the rate constants both of pore formation and of membrane permeation (or leakage) through these pores) induced by antimicrobial peptides (AMPs), such as magainin 2, and pore-forming toxins (PFTs), such as lysenin.30 Recently, we developed a new method to investigate the interaction of CPPs with lipid membranes by applying the single GUV method.31 Using this method, we can obtain the time course of entry of CPPs into single GUVs, the time course of changes in CPP concentration in the GUV membranes, and rate constant of CPP-induced pore formation in the GUV membranes.31 For example, we succeeded in demonstrating that a fluorescent probe-labeled-TP10 (CF-TP10) entered a single GUV before pore formation (before leakage of fluorescent probes), and then pores formed subsequently in the lipid membranes. In addition, we obtained information on TP10-induced pore formation, such as its rate constant and the sizes of the pores.31 These results provided valuable information on the interactions of TP10 and CF-TP10 with single GUVs. In the present study, we applied this new method to investigate the entry of oligoarginine into GUVs. We used nona-arginine (R9) because it has already been investigated thoroughly. Specifically, we simultaneously investigated


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the entry of a fluorescent probe, carboxyfluorescein (CF)-labeled R9 (CF-R9), into a GUV and the leakage of a water-soluble

fluorescence

probe,

Alexa

Fluor

647

hydrazide

(AF647),

from

single

GUVs

of

dioleoylphosphatidylglycerol (DOPG; C18:1 PG))/dioleoylphosphatidylcholine (DOPC; C18:1 PC) using confocal laser scanning microscopy (CLSM). We found that CF-R9 entered the GUVs without pore formation (i.e., without leakage of the fluorescent probe). Next, we investigated the effects of lipid composition on the entry of CF-R9 into a single GUV. It is well known that lipid membranes composed of shorter hydrocarbon chains are mechanically weak and hence smaller mechanical tension can induce rupture of the GUV due to membrane pore formation.32 Here, we used GUVs comprising dilauroyl-PG (DLPG; C12:0 PG) and ditridecanoyl-PC (DTPC; C13:0 PC) to obtain membranes containing shorter hydrocarbon chains. On the other hand, it is also well known that lipid membranes containing a high concentration of cholesterol (chol) are mechanically strong and hence higher mechanical tension is required to induce rupture of these GUVs via pore formation.33 Using the above method, we investigated the entry of CF-R9 into DLPG/DTPC-GUVs and DOPG/DOPC/chol-GUVs. On the basis of the results obtained, we discuss the elementary processes and the mechanism of entry of CF-R9 into single GUVs. ■ MATERIALS AND METHODS Materials DOPC, DOPG, DLPG, DTPC and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (PEG2K-DOPE) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Bovine serum albumin (BSA) was purchased from Wako Pure Chemical Industry Ltd. (Osaka, Japan). AF647 and N-((6-(biotinoyl) amino)-hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethyl ammonium salt (biotin-X-DHPE, referred to as biotin-lipid) were purchased from Invitrogen Inc. (Carlsbad, CA). Biotin-labeled BSA, streptavidin, and cholesterol were purchased from Sigma-Aldrich Co. (St. Louis, MO). R9 was synthesized by the FastMoc method using a 433A peptide synthesizer (PE Applied Biosystems, Foster City, CA). The sequence of R9 (9-mer) is RRRRRRRRR and it has an amide-blocked C terminus. The fluorescence probe carboxyfluorescein-labeled R9 (CF-R9) has one CF fluorophore at the N-terminus of the peptide and was synthesized using a standard method31,34 by the reaction of 5-(and 6)-carboxyfluorescein succinimidylester (Invitrogen) (11.8 mg) with R9-peptide resin (51 mg) (molar ratio reagent/peptide: 2.5/1) in dimethylformamide for 24 h at room temperature. CF-R9 was cleaved from the resin using trifluoroacetic acid (TFA), 1, 2-ethanedithiol,


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thioanisole, and MilliQ water (10/0.25/0.5/0.5, volume ratio) and phenol (0.75 g per 10 mL TFA). The method for purification of the peptide was described previously.35 The mass spectrum of CF-R9 was acquired using a MALDI-TOF-MS Autoflex (Bruker Daltonics, Billerica, MA) and α-cyano-4-hydroxy-cinnamic acid (CHCA) (Bruker Daltonics) as the matrix. The sample (1 µL) was mixed with saturated CHCA solution (4 µL) dissolved in 0.1 % (v/v) TFA in water:acetonitrile (2:1 v/v). The sample-matrix mixture (1 µL) was spotted on a polished steel target plate and crystallized at room temperature. The spectrum was measured in positive ion reflector mode. Mass calibration was performed prior to the analysis of the sample using a peptide calibration standard kit (Bruker Daltonics) to ensure accurate determination of the molecular mass. The measured mass of CF-R9 was 1780.0 ± 0.1 Da, which corresponds to the molecular mass calculated from the molecular structure. GUV preparation GUVs

of

DOPG/DOPC/biotin-lipid

DOPC/PEG2K-DOPE/biotin-lipid

(98/1/1),

(molar

ratio:

20/79/1),

DLPG/DTPC/biotin-lipid

DOPG/DOPC/biotin-lipid (20/79/1),

(40/59/1),

DLPG/DTPC/biotin-lipid

(40/59/1) and DOPG/DOPC/chol/biotin-lipid (20/59/40/1) were prepared using the natural swelling method.28 Briefly, these lipid solutions in chloroform were dried completely, then resulting dry lipid films were incubated in buffer H (10 mM HEPES, pH 7.5, 100 mM NaCl, and 1 mM EGTA) containing 0.1 M sucrose and 6 µM for AF647 at 37 °C for 2−3 h. To prepare GUVs of various lipid compositions containing small vesicles in the GUV lumen,31 first a GUV suspension was prepared in a buffer containing no AF647 using the above method, then the GUV suspension was partially purified by centrifugation at 14,000 × g for 20 min at 20 °C to remove multilamellar vesicles and lipid aggregates. A mixture of AF647 solution in buffer H containing 0.1 M sucrose and the partially purified GUV suspension was incubated with another dry lipid film at 37 °C for 2-3 h. The membrane filtering method was used to remove untrapped fluorescent probes.36 Purified GUV suspension (300 µL: 0.1 M sucrose in buffer H as the internal solution; 0.1 M glucose in buffer H as the external solution) was transferred into a hand-made microchamber.27 Single GUVs were fixed on a glass slide or a coverslip in the chamber using the strong association between biotin and streptavidin.31 First, 0.9 mg/mL BSA and 0.1 mg/mL biotin-BSA in buffer H containing 0.1 M glucose were added to the chamber and incubated. Excess biotin-BSA and BSA were removed by washing with the same buffer, leaving a BSA/biotin-BSA coating on


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the glass surface of the chamber. Next, 0.025 mg/mL streptavidin and 0.1 mg/mL BSA in the same buffer were added to the chamber and incubated, and then removed using the same buffer. Finally, a suspension of GUVs containing biotin-lipid was transferred into the chamber. Due to the strong binding of streptavidin with both the biotin-BSA adsorbed on the glass surface and the biotin-lipid in the GUV membrane, the GUVs were individually connected to the glass surface via tethers comprising a long hydrophilic segment of biotin-lipid. Investigation of the interactions of CF-R9 with single GUVs using confocal microscopy The GUVs were observed using a CLSM (FV-1000, Olympus, Tokyo, Japan) at 25 ± 1 °C using a stage thermocontrol system (Thermoplate, Tokai Hit, Shizuoka, Japan). For CLSM measurements, fluorescence images of AF647 (excited by a laser at λ = 635 nm) and of CF-R9 (excited by a laser at λ = 473 nm), and also differential interference contrast (DIC) images, were obtained using a 60× objective (UPLSAPO060X0, Olympus) (numerical aperture = 1.35).31,34 We selected GUVs containing small vesicles in the GUV lumen on the basis of their DIC images and used them to study the interaction between CF-R9 and the GUV. The probability of the GUVs containing small vesicles, judged from the DIC images, was 70% of all the GUVs prepared using the above method. To investigate the interaction of CF-R9 with a single GUV, various concentrations of CF-R9 in buffer H containing 0.1 M glucose were added continuously to the vicinity of the GUV through a 20-µm-diameter glass micropipette positioned by a micromanipulator. The distance between the GUV and the tip of the micropipette was 50 µm and the applied pressure to the micropipette, ∆P (= Pout − Pin, where Pin and Pout are the pressure in the inside and the outside of the micropipette, respectively), was −30 Pa. ∆P was measured using a differential pressure transducer (DP15, Validyne, Northridge, CA), pressure amplifier (PA501, Validyne), and a digital multimeter. Glass micropipettes were prepared by pulling 1.0 mm glass capillaries composed of borosilicate glass (G-1, Narishige, Tokyo, Japan) using a puller (PP-83 or PC-10, Narishige).27 The details of these methods were described previously.27,31,34 A special methodology was required to obtain the time course of the fluorescence intensity because the GUVs were not fixed at one position, but rather moved slightly (less than 10 µm) during the interaction with CF-R9. Consequently, to measure the time course of the fluorescence intensity of a GUV lumen due to AF647, we specified a larger region encompassing the entire area occupied by the GUV during the interaction with CF-R9 and measured


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the fluorescence intensity of this area as a function of time, then corrected this intensity by subtracting the background intensity (i.e., the fluorescence intensity of the same buffer). To measure the time course of the change in fluorescence intensity of a GUV lumen due to CF-R9, we specified a small circle ~50% of the diameter of the GUV at the center of the GUV lumen (so that the small circle did not include the GUV rim) during the interaction with CF-R9 and measured the fluorescence intensity of this area as a function of time, then corrected this intensity by subtracting the background intensity. In both cases, we were able to detect a change larger than 1% of the maximum intensity; changes less than 1% were attributed to movement of the GUV. When we measured very low fluorescence intensity using the CLSM, the intensity stabilized 40-50 s after starting the measurement due to the characteristics of the detector, so the data collected before 50 s were omitted from the analyses. To measure the time course of the fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity), a vertical line was drawn through the center of each GUV and the fluorescence intensity profile along the line was measured. The two points with the highest intensity corresponded to the GUV membrane; therefore, the mean value of the intensity of these two points was taken as the rim intensity.31 Typically, the rim intensity was measured every 5-10 s. Constant tension-induced pore formation GUVs of DLPG/DTPC (1/9) and DLPG/DTPC (4/6) (molar ratio) were prepared in buffer A (10 mM PIPES, pH 7.0, 150 mM NaCl, 1 mM EGTA) containing 0.1 M sucrose using the natural swelling method28 and the DLPG/DTPC-GUV suspensions were purified using the membrane filtering method.36 The purified GUV suspensions (300 µL) (0.1 M sucrose in buffer A as the internal solution; 0.1 M glucose in buffer A as the external solution) were individually transferred into the hand-made microchamber mentioned above.27 The GUVs were observed using an inverted fluorescence phase-contrast and DIC microscope (IX-71, Olympus) at 25 ± 1 °C using a stage thermocontrol system (Thermoplate, Tokai Hit). Phase-contrast and DIC images of GUVs were recorded using a CCD camera (CS230B, Olympus) equipped with a hard disk. To obtain the rate constant for constant tension-induced pore formation in a GUV, we used the same method previously developed by us.37 Tension was applied to the lipid membrane of single GUVs using the micropipette aspiration method.38 A micropipette was coated with 0.5% (w/v) BSA in buffer A containing 0.1 M glucose, then a single GUV was held at the tip of the micropipette by applying a suction pressure, ∆P. The GUV was observed until it was completely aspirated into the micropipette as a result of pore formation. The time of pore formation was


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Biochemistry

defined as the time when the GUV was completely aspirated and this measurement had a resolution of less than 1 s. The tension of the GUV membrane, σ, can be described as a function of ∆P as follows:38

σ=

∆Pd P 4(1 − d P / DV )

(1)

where dp is the internal diameter of the micropipette and Dv is the diameter of the spherical part of the GUV exterior to the micropipette. ■ RESULTS AND DISCUSSION Entry of CF-R9 into single DOPG/DOPC-GUVs without pore formation We first investigated the interaction of CF-R9 with single DOPG/DOPC/biotin-lipid-GUVs (molar ratio, 20/79/1, hereinafter abbreviated DOPG/DOPC (2/8)) containing AF647 and small vesicles 1-10 µm diameter composed of 20%DOPG/80%DOPC suspended inside the GUV (i.e., the GUV lumen). Figure 1A shows a typical experimental result of the interaction of 10 µM CF-R9 with a single GUV. The CF-R9 solution was continuously provided to the vicinity of the GUV through a micropipette; consequently, the CF-R9 concentration near the GUV approached steady state and was almost the same as the concentration inside the micropipette.30 The fluorescence intensity of AF647 in the GUV lumen remained essentially constant during the addition of the 10 µM solution of CF-R9 (up to 6 min) (Figure 1A (1) and red curve in Figure 1B), showing that no leakage of AF647 occurred. This result indicates that CF-R9 did not induce pores in the lipid membrane through which AF647 leaked. It is noted that the small vesicles in the GUV lumen did not contain AF647 due to the method used to prepare the GUVs, so the fluorescence intensities of AF647 at the positions of the small vesicles were a little smaller (Figure 1A (1)). Figure 1A (2) shows that the fluorescence intensity of the GUV membrane (the rim intensity) due to CF-R9 gradually increased and became essentially steady at 25 s (green squares in Figure 1B). At the beginning of the interaction, there was no fluorescence inside the GUV, but after 33 s, fluorescence intensity was observed in the membranes of the small vesicles in the GUV lumen (t = 33−248 s in Figure 1A(2)). This fluorescence occurred without pore formation. These results indicate that CF-R9 entered the GUV lumen from the outside by translocating across the GUV membrane and then bound to the membranes of the small vesicles in the GUV lumen without pore formation. This experiment was repeated with 21 GUVs and similar results were obtained. The entry of CF-R9 into the GUV lumen within 6 min interaction of CF-R9 with a single GUV was observed with 11 GUVs, and hence the fraction of


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entry of CF-R9 at 6 min without pore formation, Pentry (6 min), was 0.52. We confirmed the reproducibility of Pentry by conducting two independent experiments using 17-21 GUVs and obtained similar results. The mean value and the standard error of Pentry (6 min) was 0.47 ± 0.06. We also investigated the concentration dependence of the entry of CF-R9 into a GUV without leakage of AF647. At CF-R9 concentrations of ≤ 5.0 µM, Pentry (6 min) = 0, and at and above 7.0 µM CF-R9, Pentry (6 min) increased with an increase in CF-R9 concentration and was 0.90 at 28 µM (green □ in Figure 1C). To elucidate the effects of the surface charge density on the entry of CF-R9 into a GUV, we investigated the interaction of CF-R9 with single GUVs of electrically neutral DOPC/PEG2K-DOPE/biotin-lipid (molar ratio, 98/1/1, hereinafter called DOPC-GUVs) containing small DOPC vesicles and AF647 in the GUV lumen. PEG2K-DOPE was incorporated into the GUV membrane to induce the formation of electrically neutral DOPC-GUVs in buffer H containing high concentration of salts.39 Figure S1 in the Supporting Information (SI) shows a typical experimental result of the interaction of 35 µM CF-R9 with a single DOPC-GUV. No fluorescence intensity due to the membranes of the small vesicles in the GUV lumen was detected during the 6 min interaction, indicating that no entry of CF-R9 occurred (Figure S1 (2)). Moreover, the rim of the GUV was not detected as a peak in the fluorescence intensity profile shown in Figure S1 (2), indicating that the CF-R9 concentration in the GUV membrane was low. In other GUVs we found no entry of CF-R9 and no rim intensity under this condition (two independent experiments each using 15 GUVs). Therefore, Pentry (6 min) = 0 for DOPC-GUVs (red ∆ in Figure 1C). On the other hand, CF-R9 entry into DOPG/DOPC/biotin-lipid (40/59/1)-GUVs (hereinafter called DOPG/DOPC (4/6)-GUVs) was observed (Figure S2). Pentry (6 min) for DOPG/DOPC (4/6)-GUVs increased with CF-R9 concentration, and it was larger than Pentry (6 min) for DOPG/DOPC (2/8)-GUVs at the same CF-R9 concentration (blue ○ in Figure 1C). These results indicate that the rate of entry of CF-R9 into a GUV increased as the surface charge density increased. To elucidate the kinetics of entry of CF-R9, we next investigated and analyzed the time course of the increase in CF-R9 concentration in the GUV membrane, CM (t) [M], using the same method as in our previous paper.31 We investigated the interaction of CF-R9 with a single GUV that did not contain small vesicles because the presence of small vesicles in the GUV lumen (Figure 1) near the GUV membrane caused fluctuations in the fluorescence intensity of the GUV membrane.31 It is difficult to analyze the rapid increase in rim intensity shown in Figure 1B


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Biochemistry

using the present method,30,31 so we used lower concentrations of CF-R9. Figure 2A shows a typical result of the interaction of 5.0 µM CF-R9 with a single DOPG/DOPC (2/8)-GUV containing AF647. Figure 2A (1) and 2B indicate that no leakage of AF647 occurred up to 6 min. Figure 2A (2) shows that the rim intensity gradually increased with time and at 200 s was essentially steady (green squares in Figure 2B). On the other hand, the fluorescence intensity of the GUV lumen due to CF-R9 did not increase at 6 min. We also investigated the interaction of 40 µM CF-R9 with DOPG/DOPC (4/6)-GUVs and found that no leakage of AF647 occurred and the fluorescence intensity of the GUV lumen due to CF-R9 did not increase at 6 min, although 20% of the GUVs had burst. The fraction of entry was 1.0, but the CF-R9 concentration in the GUV lumen at 6 min was less than 1% of the possible maximum concentration, which is the concentration of CF-R9 outside the GUV. We previously proposed a series of processes to describe the entry of CF-TP10 into single GUVs,31 and here we propose the same processes to describe the entry of CF-R9 into a GUV lumen (Figure 3). First, CF-R9 in the bulk solution binds at the membrane interface of the outer monolayer of the GUV at a rate constant kON. Next, CF-R9 transfers from the outer monolayer to the inner monolayer at a rate constant kFF, and then transfers from the inner monolayer into the lumen adjacent to the GUV membrane (where the CF-R9 concentration is Cin) at a rate constant kOFF. Finally, CF-R9 diffuses into the GUV bulk lumen at a rate constant kdiff. It is also necessary to consider the backward reactions. After starting the addition of CF-R9 solution from the micropipette, the CF-R9 concentration in the vicinity of a GUV increases from zero to a constant, steady value, C eq [M], for a short time, and remains out constant during the interaction of CF-R9 with the single GUV.30,34 Consider the case where the transfer of CF-R9 between the outer and inner monolayers is fast, i.e., the rate of the transfer is faster than the rate of binding of CF-R9 and faster than the rate of unbinding from the membrane to aqueous solution. For this case, the following equations hold true and the concentration of CF-R9 bound to the GUV membrane, CM (t), is obtained for the initial time (i.e., when the CF-R9 concentration in the GUV lumen is low):31

CM (t ) = A [1 − exp(− kappt )]

(2)

eq where kapp = kONCout / 2 + kOFF

(3)

where kapp is the apparent rate constant of the increase in CM (t) and A is a constant. The time course of the increase in the rim intensity of DOPG/DOPC (2/8)-GUVs (Figure 2B) was fit well by eq. 2 (the black line in Figure 2B) and


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gave a value for kapp of 1.6 × 10−2 s−1. Figure 2C shows that kapp increased linearly with an increase in C eq , which out agrees with eq. 3, and the data fit with eq. 3 provided values of kON = (4.2 ± 0.3) × 103 M−1s−1 and kOFF = (5.7 ± 0.8) × 10−3 s−1. Therefore, the binding constant of CF-R9 to the membrane, KB, is determined by KB =kON/kOFF: hence KB = (7.0 ± 1.0) × 105 M−1 for DOPG/DOPC (2/8)-GUVs. We obtained similar results for DOPG/DOPC (4/6)-GUVs and fitting the data to eq. 3 in Figure 2C provided kON = (1.2 ± 0.1) × 104 M−1s−1 and kOFF = (9.0 ± 1.0) × 10−3 s−1; therefore, KB = (1.3 ± 0.2) × 106 M−1. The values for kON, kOFF, and KB for DOPG/DOPC (4/6)-GUVs were larger than those for DOPG/DOPC (2/8)-GUVs. The KB of CF-R9 to the DOPG/DOPC (2/8) membrane ((7.0 ± 1.0) × 105 M−1) is larger than the KB of R9 to 1-palmitoyl-2-oleoyl-PG (POPG)/1-palmitoyl-2-oleoyl-PC (POPC) (75/25) membrane (8.2 ×104 M−1),15 and this higher KB is due to the hydrophobic probe (CF) attached to R9 in CF-R9. Effects of the hydrocarbon chain length of lipids on the entry of CF-R9 into single GUVs The effect of hydrocarbon chain length on the mechanical properties of lipid membranes were elucidated by investigating the effect of constant tension on DLPG/DTPC (1/9)-GUVs using a method recently developed by us .37,40 We applied constant tension to a GUV in buffer A using a micropipette for a specific time and observed its rupture. We initially held a single GUV at the tip of a micropipette for a few minutes using only slight aspiration pressure, thus providing a tension on the bilayer of ~0.5 mN/m, and then rapidly (~10 s) increased the aspiration pressure to a tension (σ) of 3.0 mN/m and held this tension for a specific time. After a period of time, the GUV was suddenly aspirated into the micropipette. This can be explained by first a pore forming in the GUV membrane, causing rupture of the GUV, and then the GUV being completely aspirated into the micropipette due to the pressure difference between the inside and the outside of the micropipette. When we repeated the same experiment using 20 DLPG/DTPC (1/9)-GUVs, we found that pore formation occurred stochastically at different times. The time course of the fraction of intact GUVs among all of the examined GUVs, Pintact(t), was well fit by a single exponential decay function as follows (Figure 4A):

Pintact (t ) = exp(− k p t )

(4)

where kp is the rate constant of the tension-induced rupture of a GUV, which is the same as the rate constant of tension-induced pore formation, and t is the time that the constant tension was applied to a GUV (constant tension


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Biochemistry

was applied at t = 0). From this fitting, we obtained a value of kp of 1.4 × 10−2 s−1. Next, we performed the same experiment (n = 20) using σ = 1.0 mN/m. At this constant tension, the decrease in Pintact (t) with time was slower than the decrease at 3.0 mN/m, and kp obtained from curve fitting was 1.8 × 10−3 s−1. The kp values for DLPG/DTPC (1/9)-GUVs (□) and of DLPG/DTPC (4/6)-GUVs (○) increased greatly with tension, and the tensions required to induce pore formation in DLPG/DTPC (4/6)-GUVs were smaller than those for DLPG/DTPC (1/9)-GUVs at similar kp values (Figure 4B). This can be reasonably explained by the electrostatic interaction effect.40 For comparison, the data for kp for DOPG/DOPC (1/9)-GUV and DOPG/DOPC (4/6)-GUV are also shown in Figure 4B. The tensions required to induce pores in DLPG/DTPC-GUVs were smaller than those in DOPG/DOPC-GUVs, indicating that DLPG/DTPC-GUVs are mechanically weaker than DOPG/DOPC-GUVs. Next, we investigated the interaction of CF-R9 with GUVs of DLPG/DTPC/biotin-lipid (20/79/1) (hereinafter called DLPG/DTPC (2/8) GUVs) containing AF647 and small vesicles composed of 20%DLPG/80%DTPC in the GUV lumen. This membrane has almost the same surface charge density as that of DOPG/DOPC (2/8) membrane. CF-R9 at 5.0 µM did not induce leakage of AF647 up to 6 min (Figure 5A (1)). After 62 s of interaction, fluorescence intensity was observed from the membranes of the small vesicles in the GUV lumen (t = 62−360 s) (Figure 5A (2)). At and above 5.0 µM CF-R9, Pentry (6 min) increased with an increase in CF-R9 concentration, and was 1.0 at 28 µM (Figure 5C). At the same concentrations of CF-R9, the values of Pentry (6 min) for single DLPG/DTPC (2/8)-GUVs were larger than those for DOPG/DOPC (2/8)-GUVs. We also investigated and analyzed the kinetics of entry of CF-R9 into single DLPG/DTPC (2/8)-GUVs and the time course of the increase in the CF-R9 concentration in the GUV membrane (Figure S3). The time course of the rim intensity (Figure S3B) was fit well by eq. 2 and gave a value for the rate constant kapp of 2.3 ×10−2 s−1. Figure S3C shows that kapp increased linearly with an increase in C eq and fitting the data to eq. 3 in Figure S3C provided out the values kON = (7.2 ± 0.6) ×103 M−1s−1 and kOFF = (7.3 ± 1.0) ×10−3 s−1. Therefore, KB = (1.0 ± 0.2) ×106 M−1. The values of kON and KB of CF-R9 for DLPG/DTPC (2/8)-GUVs were slightly larger than those of DOPG/DOPC (2/8)-GUVs, but the values of kOFF for both GUVs were essentially the same within experimental error. Generally, the binding of peptides with a membrane interface is determined by both electrostatic interactions and interfacial hydrophobicity.41 The surface charge density of the DLPG/DTPC (2/8) membrane is almost the same as that of the


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DOPG/DOPC (2/8) membrane, and hence the effect of electrostatic interactions on binding should be similar. The hydrocarbon chains of lipids in the membrane interface determine the membrane interfacial hydrophobicity.41 The free energy of the transfer of hydrocarbon containing a double bond from hydrocarbon liquid to water is smaller than that of saturated hydrocarbons,42 suggesting that the distribution of hydrocarbon chains at the membrane interface of DLPG/DTPC (2/8) with no double bond is different from that of DOPG/DOPC (2/8) with double bonds. This difference in membrane interfacial hydrophobicity may cause the difference in the binding of CF-R9 to these membranes. Finally, we investigated the interaction of CF-R9 with DLPG/DTPC (4/6)-GUVs not containing small vesicles (Figure 6). The fluorescence intensity of the GUV lumen due to AF647 remained essentially constant during the addition of 40 µM CF-R9 for the first 170 s, then the fluorescence intensity gradually decreased (Figure 6A (1) & the red curve in Figure 6B). At 360 s, the fluorescence intensity was 27% of the initial intensity (at t = 0), although a fluorescence microscope image of the same GUV showing CF-R9 fluorescence (Figure 6A (2)) indicates that the GUV was intact and spherical. As discussed in our reports on AMPs and PFTs,26-30 the decrease in fluorescence intensity results from the leakage of AF647 from the GUV through CF-R9-induced pores in the membrane. Thus, the time at which the fluorescence intensity began to rapidly decrease (t = 170 s) corresponds to the time when pores were formed in the membrane. On the other hand, the fluorescence intensity of the GUV lumen due to CF-R9 rapidly increased with time after pore formation (Figure 6A (2) & the blue curve in Figure 6B). It appears that the rapid and substantial entry of CF-R9 into the GUV lumen occurred by the permeation of CF-R9 through the pores. The fraction of leaked GUVs (i.e., the fraction of GUVs with pores) of all the examined GUVs (n = 20) at 6 min was 0.55, although 20% of the total GUVs had burst. On the other hand, the slight increase in the fluorescence intensity of the GUV lumen due to CF-R9 occurred before pore formation, and its intensity and time course greatly depended on the GUV. This slight increase is likely due to the fluorescence of small vesicles inside the GUV lumen and these vesicles were produced spontaneously using the standard preparation method of GUVs that do not contain vesicles. The amount of spontaneously generated small vesicles in DLPG/DTPC (4/6)-GUVs was much larger than in other GUVs. Figure 6C shows the CF-R9 concentration dependence of the fraction of leaked GUVs: at 20 µM it was almost 0, but it increased with increasing CF-R9 concentration. We also investigated the interaction of CF-R9 with DLPG/DTPC (4/6)-GUVs containing small vesicles in their lumens by conducting two independent


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Biochemistry

experiments each using 15 GUVs, and obtained Pentry (6 min). At 5.0 µM CF-R9, Pentry (6 min) = 0.50, and at 20 and 40 µM, the Pentry (6 min) values were 1.0 (Figure 6C). In Figure 6B, at 20 s before the start of membrane permeation of AF647, the fluorescence intensity of the GUV lumen due to CF-R9 and also the rim intensity started to increase rapidly. This result may be explained by a mechanism similar to that proposed for magainin 2-induced pore formation.34 At the beginning of pore formation, the pore radius is initially zero and then increases, so initially the radius of the pore is too small for AF647 to pass through the pore and thus no leakage occurs. When the pore size becomes larger than the diameter of CF-R9, the CF-R9 molecules bound to the outer monolayer can diffuse into the inner monolayer via the wall of the toroidal structure of the pore43-45 and the CF-R9 molecules transfer to the GUV lumen from the inner monolayer. Effects of cholesterol on the entry of CF-R9 into single GUVs To elucidate the effects of cholesterol on the entry of CF-R9 into a GUV, first we investigated the interaction of CF-R9 with single GUVs of DOPG/DOPC/chol/biotin-lipid (20/59/40/1, molar ratio) (hereinafter called DOPG/DOPC/chol (2/6/4)). The cross-sectional area of DOPC under no tension,46 Ao, is 0.73 nm2, and we assume that Ao is the same for both DOPG and DOPC. Ao of cholesterol in DOPC membranes47 was estimated to be 0.35-0.40 nm2, so the Ao of cholesterol is about half that of DOPC or DOPG. Based on these values, we can conclude that the surface charge density of DOPG/DOPC/chol (2/6/4) is almost the same as that of DOPG/DOPC (2/8). The cholesterol concentration in the DOPG/DOPC/chol (2/6/4) membrane is 33 mol%, which is relatively high compared to biomembranes.48 Figure 7A shows the effect of the interaction of 28 µM CF-R9 with single DOPG/DOPC/chol (2/6/4)-GUVs containing AF647 and small vesicles composed of DOPG/DOPC/chol (2/6/4) in the GUV lumen. No fluorescence intensity was observed from the membranes of the small vesicles in the GUV lumen during the 6 min interaction. We conducted two independent experiments on a total of 36 GUVs (nt = 36) and no entry of CF-R9 was observed under this condition. Having the concentration of CF-R9 to 14 µM provided the same results. We next determined the time course of the increase in CF-R9 concentration in the GUV membrane. Figure S4 shows the effect of the interaction of 5.0 µM CF-R9 with single DOPG/DOPC/chol (2/6/4)-GUVs containing AF647 but not containing small vesicles, and Figure 7B shows these results graphically: the rim intensity gradually increased and at 200 s it was essentially steady state. The rim intensity at steady state for DOPG/DOPC/chol (2/6/4)-GUVs was almost half


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that of DOPG/DOPC (2/8)-GUVs at 5.0 µM CF-R9 (Figure 7B). The ratio of the rim intensity of DOPG/DOPC (2/8)-GUVs at steady state (I2) to that of DOPG/DOPC/chol (2/6/4) (I1), (I2/I1), was 1.9 ± 0.1 (nt = 30). For 10 µM CF-R9, I2/I1 = 1.95 ± 0.05 (nt = 29). As discussed above, the CF-R9 concentration in the membrane, CM (t), is proportional to the rim intensity. Therefore, the above results indicate that CF-R9 is present only in the outer monolayer of DOPG/DOPC/chol (2/6/4) but in both monolayers of DOPG/DOPC (2/8)-GUVs, showing that the transfer of CF-R9 from the outer to the inner monolayer of DOPG/DOPC/chol (2/6/4) was essentially inhibited and kFF ≈ 0. Hence, CM (t) is approximately equal to the CF-R9 concentration in the outer monolayer, COM. The equation describing CM (t) for these conditions was derived using a similar method31 and is provided in SI. The equation describing CM (t) (eq. S3) is the same as eq. 3, whereas the equation describing kapp (eq. S4) is slightly different from eq. 4. The time course of the change in the rim intensity of the DOPG/DOPC/chol (2/6/4)-GUVs (Figure 7B) was fit well by eq. S3 and provided a value for kapp of 1.5 × 10−2 s−1. Figure 7C shows that kapp increased linearly with an increase in C eq , and out fitting the data to eq. S4 provided the values kON = (2.37 ± 0.06) × 103 M−1s−1 and kOFF = (1.1 ± 0.4) ×10−3 s−1. Hence, KB = (2.4 ± 0.9) × 106 M−1, which is larger than the KB value for DOPG/DOPC (2/8) membranes. Within experimental error, the value of kON for the membrane containing cholesterol was approximately half that of the membrane without cholesterol. Since CM (t) = (COM + CIM)/2 (where CIM is the CF-R9 concentration in the inner monolayer), if we consider CIM = 0, then we can reasonably explain the result obtained for DOPG/DOPC/chol (2/6/4)-GUVs. Therefore, this result supports the conclusion that CF-R9 is present only in the outer monolayer of DOPG/DOPC/chol (2/6/4)-GUVs. ■ GENERAL DISCUSSION The results presented in this report show that CF-R9 translocates continuously across the lipid membranes of single DOPG/DOPC (2/8)-GUVs, DOPG/DOPC (4/6)-GUVs, and DLPG/DTPC (2/8)-GUVs, and then enters their lumens without pore formation (judged from the absence of leakage of AF647) within the experimental timeframe (6 min). These results are in contrast to our previous results obtained using CF-TP10, in which we demonstrated that CF-TP10 enters a single GUV before pore formation and then pore formation occurs subsequently in the lipid membranes. Here we consider the elementary processes leading to entry (Figure 3) to elucidate a factor determining


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the rate of the entry of CF-R9 into GUVs without pore formation, Ventry.31 The CF-R9 concentration in the membrane of the small vesicles in the GUV lumen increases with an increase in the CF-R9 concentration in the aqueous solution within the GUV lumen, Clumen. Hence, Ventry or Pentry (6 min) is determined by the rate of increase in Clumen, dClumen/dt, which increases with Cin. At the initial time, dCin/dt ≈ kOFFCIM (see eq. 4 in ref. 31). Since the values of kOFF of CF-R9 for both DOPG/DOPC (2/8) and DLPG/DTPC (2/8) GUVs are similar, Ventry is determined by CIM. dCIM/dt is mainly determined by kFFCOM for the initial time (see eq. 3 in ref. 31). The rate of increase in COM is

mainly determined by kON for the initial time (see eq. 2 in ref. 31). Therefore, when kFF and/or kON are large, Cin rapidly increases with time, and hence Pentry (6 min) of CF-R9 becomes larger. For example, the values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8)-GUVs are larger than those of DOPG/DOPC (2/8)-GUVs for the same CF-R9 concentrations (Figure 5C). This result can be explained by the larger values of kFF and/or kON for DLPG/DTPC membranes. On the other hand, the result of Pentry (6 min) = 0 for DOPG/DOPC/chol (2/6/4)-GUVs can be explained by kFF ≈ 0. In contrast, the result of Pentry (6 min) = 0 for DOPC-GUVs can be explained by kON ≈ 0. Next we consider which factor determines the value of kFF in GUV membranes. As described in the Results section, CF-R9 did not induce pore formation in most membranes. Here, we define a pore as a water channel in a lipid membrane that is always open and whose diameter is sufficiently large so that fluorescent probes such as AF647 can pass through the pore. However, the structures of lipid membranes in the liquid-crystalline phase are believed to fluctuate considerably, resulting in temporal decreases in the lateral density of lipid membranes.37 Each area of decreased density is treated as a pre-pore with a certain effective size, which can be approximated as a circle with radius r. The probability of the occurrence of a pre-pore increases in a stretched membrane or when tension is applied. According to the classical theory of tension-induced pore formation,49-51 once such a pre-pore is formed in the membrane, the total free energy of the system changes by an additional free energy component (called the free energy of a pre-pore, U (r)) that consists of two terms: one (−πr2σ) that is associated with lateral tension (σ), favoring expansion of the pre-pore, and another (2πrΓ) that is associated with the line tension (Γ) (i.e., the line free energy per unit length) of the pre-pore edge, favoring pre-pore closure. The free energy of a pre-pore, U (r), can therefore be expressed as follows:

U (r ) = 2πrΓ − π r 2σ

(5).


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U (r) has a maximum of Umax = U (r*) = πΓ2/σ at r = r* (= Γ/σ). If the radius of a pre-pore is less than the critical

radius, r*, it closes quickly. However, if the radius expands and reaches r*, the pre-pore transforms into a pore. It is noted that the radius of the pre-pores is much smaller than that of the pore. This classical theory has been used to explain several phenomena, such as the rate of closure of pores33,52 and the tension dependence of rate constant of tension-induced pore formation.32,37,40,53 In the case of CF-R9, pore formation does not occur, but pre-pores are formed due to thermal fluctuation of the lipid membranes. We suggest the following hypothesis to describe the mechanism of entry of CF-R9 into a GUV lumen. Binding of CF-R9 to the membrane interface of the outer monolayer induces tension in the membrane and the membrane stretches, which increases the formation of pre-pores in the membrane. CF-R9 can translocate from the outer to the inner monolayer by passing through these pre-pores. In our previous paper,34 we demonstrated experimentally that the binding of an antimicrobial peptide, magainin 2, to the membrane interface of the outer monolayer of a GUV increases the area of the GUV membrane, which induces tension in the membrane, resulting in the membrane stretching. Above the critical tension, magainin 2 induced pore formation in the lipid membrane, through which AF647 in the lumen of the GUV leaked out. However, prior to pore formation, magainin 2 was unable to translocate from the outer to the inner monolayer.34 Recently Sun et al. presented molecular dynamics simulation results indicating that octaarginine (R8) can hinder the closing of membrane pores and proposed that R8 can pass through naturally occurring thermal pores.54 These pores correspond to the pre-pores mentioned above. Therefore, we suggest that CF-R9 can hinder the closing of pre-pores and translocate from the outer to the inner monolayer by passing through the pre-pores. It was reported that R9 induced small, fluctuating electric currents in a planar membrane when voltage was applied across the membrane.55 These currents are not due to a stable pore, but rather to transient pre-pores with small radii because ion translocation through the pre-pores induces these currents. Therefore, this result may support the above hypothesis. When we consider the difference in the results obtained using CF-R9 and magainin 2 (namely, that CF-R9 can translocate through pre-pores but magainin 2 cannot), the effect of peptide-pre-pore interactions on the stability of the pre-pore may play an important role in the translocation of peptides through pre-pores without the formation of pores. Figure 8 shows a free energy profile of a pre-pore, U (r), for different line tensions, Γ. Umax of the free energy of a pre-pore decreases with a decrease in Γ, since Umax = πΓ2/σ. The initial slope of U (r) decreases with a


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Biochemistry

decrease in Umax, so the pre-pore radius corresponding to U (r) = 5 kT increases with a decrease in Γ (e.g., r = 0.2, 0.3, and 0.7 nm for Γ = 18, 12, and 6 pN, respectively) (Figure 8). This indicates that the radii of pre-pores formed by thermal fluctuations increase with a decrease in Γ. It is reported that Γ greatly decreases with a decrease in the chain length of lipid molecules.32 For example, Γ of DTPC (C13:0) is 6.2 pN (U (r) of a DTPC membrane corresponds to curve A in Figure 8), but Γ of DOPC (C18:1) is 11.5 pN (U (r) of a DOPC membrane corresponds to curve B in Figure 8).32 Therefore, we can reasonably consider that the frequency of pre-pore formation and the size of pre-pores in DLPG/DTPC (2/8)-GUVs are larger than those of DOPG/DOPC (2/8)-GUVs. On the other hand, Γ increases with an increase in cholesterol concentration; for example, the presence of 20 mol% cholesterol in DOPC-GUVs increases the Γ value by 5.3 pN.33 Hence, we can expect that the Γ value of DOPG/DOPC/chol (2/6/4) is larger than that of DOPG/DOPC (2/8) by more than 5.3 pN (U (r) of a DOPC/chol membrane corresponds to curve C in Figure 8), indicating that the initial slope of U (r) is very large. Therefore, it is likely that in DOPG/DOPC/chol (2/6/4)-GUVs, the frequency of pre-pore formation is low and the size of the pre-pores is smaller than that of the CF-R9 molecule, and this suppresses the entry of CF-R9 into DOPG/DOPC/chol (2/6/4)-GUVs. On the basis of this analysis, we conclude that the size of the pre-pores and the frequency of pre-pore formation greatly depend on the mechanical properties of the lipid membranes as represented by the line tension, and that therefore the rate of entry of CF-R9 into a GUV lumen may likewise greatly depend on the mechanical properties of lipid membranes. As described above, CF-R9 entered DOPG/DOPC-GUVs or DLPG/DTPC (2/8)-GUVs without pore formation, as determined by the absence of leakage of the fluorescent probe in the lumen. However, the amount of CF-R9 that entered into the GUV lumen was small: the CF-R9 concentration in the GUV lumen at 6 min was less than 1% of the expected maximum concentration. These results indicate that our experimental method31 detects the entry of peptides into a GUV lumen with high sensitivity. On the other hand, it is reported that R6 and Tat peptide induced pore formation in membranes composed of specific lipids such as DOPS and DOPE, and concomitantly these peptides entered the GUVs through the pores.22,25,56 Fluorescent probes can leak through the pore continuously, as is the case for pores induced by AMPs and PFTs30. For example, R6-FITC and rhodamine (Rh)-labeled Tat peptide induced complete leakage of fluorescent probes such as AF633 from GUVs comprising high concentrations of DOPS and DOPE, indicating pore formation, and subsequently R6-FITC or Rh-Tat entered the GUVs.22,25


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Ciobanasu et al. also indicated that fluorescent probe-labeled Tat peptide (Tat (48-57)) induced substantial leakage of AF647 from DOPS/DOPC/chol- GUVs and DOPS/DOPE/chol-GUVs, indicating that pores had formed and that Tat peptides passed through the pores and entered the GUVs.56 These results indicate that R6 and Tat peptide induced pore formation in lipid membranes containing DOPS and/or DOPE, and then substantial amounts of these peptides entered the GUVs through the pores, similar to the entry of CF-R9 into DLPG/DTPC (4/6)-GUVs described in the present report. However, the rate and the amount of entry of peptides into the lumens of these GUVs were much higher than those of CF-R9 into DOPG/DOPC-GUVs. This can be explained as follows. If CPPs can induce pores in membranes, the rate of entry of CPPs into the GUV lumen is high because CPPs can pass through the pores. In contrast, if CPPs cannot induce pores, the rate of entry of CPPs into the GUV lumen is small because CPPs must pass through the transient, short-lived pre-pores. It was recently reported that the AMP Polybia-MP1 can induce pore formation in GUVs containing DOPE and palmitoyl-oleoyl-phosphatidylserine (POPS).57 Therefore, we can consider that peptide-induced pore formation in lipid membranes greatly depends on the constituent lipids and the buffer conditions (e.g., salt concentration and pH). To elucidate the mechanism of entry of peptides in these systems, it is necessary to investigate the mechanism of peptide-induced pore formation. As described in the introduction, the generation of negative Gaussian curvature may explain pore formation due to the specific interaction of peptides with lipids.22,25 The results of Figure 4 show that DLPG/DTPC (4/6)-GUVs are the mechanically weakest GUVs; therefore, pore formation occurred easily. This explains why pore formation occurred only in these GUVs among all the GUVs examined in this report. It is well known that AMPs induce pore formation in the bacterial cell membrane and thus have a bactericidal effect.28,34,58,59 Therefore, understanding the mechanism of AMP-induced pore formation would help elucidate the mechanism of CPP-induced pore formation. In conclusion, the reaction scheme of CPPs with lipid membranes depends on their lipid composition. Further studies are thus necessary. ■ CONCLUSION The results provided in this report show that there are two modes of entry of CF-R9 into a GUV lumen. In mode A, CF-R9 translocates continuously across the lipid membrane of single DOPG/DOPC (2/8)-, DOPG/DOPC (4/6)-, and DLPG/DTPC (2/8)-GUVs and enters these GUVs without pore formation (i.e., without the leakage of fluorescent probes trapped in the lumen of the GUV), but the amount of CF-R9 entering the GUV lumen is small. In contrast, in mode B, CF-R9 enters the lumen of a DLPG/DTPC (4/6)-GUV through pores and the rate of entry of the


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Biochemistry

peptide and amount of peptide entering the GUV lumen are much larger than in mode A. The major characteristic of mode A is that Ventry greatly depends on the lipid composition of the GUV: for example, Ventry for DLPG/DTPC-GUVs is larger than that for DOPG/DOPC-GUVs, and Ventry for DOPG/DOPC/cholesterol (2/6/4)-GUVs is 0. These results suggest that Ventry increases with a decrease in the mechanical strength of the lipid membrane. Analysis of the elementary processes underlying the entry of CF-R9 into GUVs indicates that kFF is a main factor determining Ventry. We propose that CF-R9 can translocate from the outer to the inner monolayer of a GUV by passing through pre-pores. This hypothesis is consistent with the dependence of the rate of entry of CF-R9 into GUVs on the mechanical stability of the lipid membrane. Supporting Information Available Figures of leakage of AF647 and entry of CF-R9 into single DOPC-GUVs and DOPG/DOPC (4/6)-GUVs containing small vesicles, time course of the increase in rim intensity due to CF-R9 in single DLPG/DTPC (2/8)-GUVs, DOPG/DOPC/chol (2/6/4)-GUVs not containing small vesicles, and derivation of CM (t) in DOPG/DOPC/chol (2/6/4)-GUV REFERENCES (1) Magzoub, M., and Gräslund, A. (2004) Cell-penetrating peptides: small from inception to application. Quart. Rev. Biophys. 37, 147-195.

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Bax-derived peptide: Evidence for lipidic pores, Proc. Natl. Acad. Sci. USA., 105, 17379-17383. Figure Legends Figure 1: Leakage of AF647 and entry of CF-R9 into single DOPG/DOPC-GUVs containing small vesicles, induced by CF-R9. (A) CLSM images of (1) AF647, (2) CF-R9, and (3) DIC in a DOPG/DOPC (2/8)-GUV for the interaction of 10 µM CF-R9 with the GUV. The numbers above each image show the time in seconds after the addition of CF-R9 was started. The bar corresponds to 20 µm. (B) Time course of the increase in the normalized fluorescence intensity of the GUV shown in (A). Red and green points correspond to the fluorescence intensity of the GUV lumen due to AF647 and that of the GUV rim due to CF-R9, respectively. (C) Dependence of Pentry (6


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min) without pore formation on the CF-R9 concentration. DOPG/DOPC (2/8)- (green □), DOPG/DOPC (4/6)- (blue ○), and DOPC-GUVs (red ∆). Figure 2: Time course of rim intensity due to CF-R9 in single DOPG/DOPC-GUVs not containing small vesicles induced by CF-R9. (A) CLSM images of (1) AF647 and (2) CF-R9 in a DOPG/DOPC (2/8)-GUV for the interaction of 5.0 µM CF-R9. The numbers above each image show the time in seconds after the addition of CF-R9 was started. The bar corresponds to 20 µm. (B) Time course of the increase in the normalized fluorescence intensity of the GUV shown in (A). Red and green points correspond to the fluorescence intensity of the GUV lumen due to AF647 and that of the GUV rim due to CF-R9, respectively. The solid black line represents the best fit curve using eq. 2. The obtained value of kapp was 0.016 s-1. (C) Dependence of kapp on CF-R9 concentration. The mean values and standard errors of kapp are shown. (□) DOPG/DOPC (2/8)- and (green ○) DOPG/DOPC (4/6)-GUVs. The solid red line represents the best fit curve using eq. 3. Figure 3: A scheme of the elementary processes of the entry of CF-R9. Clumen, Cin, and C eq are CF-R9 out concentration in the GUV bulk lumen, in the GUV lumen adjacent to the membrane, and in aqueous solution outside the GUV adjacent to the membrane. COM and CIM are CF-R9 concentration in the outer and inner monolayer of the GUV, respectively. kON, kOFF, and kdiff are rate constant of the binding of CF-R9 to the monolayer of a GUV from aqueous solution, of unbinding of CF-R9 from the monolayer to the aqueous solution adjacent to the membrane, and of the diffusion from the GUV lumen adjacent to the membrane to the bulk lumen (i.e., the central region of the GUV). kFF is the rate constant of the translocation of CF-R9 from one monolayer to the other one. Figure 4: The effect of constant tension on rupture of single GUVs. (A) Time-course of the fraction of intact DLPG/DTPC (1/9)-GUVs without rupture among all of the examined GUVs, Pintact(t), in the presence of tension; σ: (○) 1.0, (□) 3.0 mN/m. The number of the examined single GUVs was 20 in each experiment. The solid line represents the best fit curve of eq. 4. (B) Dependence of kp on tension. (□) DLPG/DTPC (1/9)-GUV, (○) DLPG/DTPC (4/6)-GUV, (■) DOPG/DOPC (1/9)-GUV, and (●) DOPG/DOPC (4/6)-GUV. Mean values and standard errors of kp for each tension were determined among three independent experiments using 20 GUVs for each experiment. Error bars show standard errors. The data for DOPG/DOPC (4/6) are reprinted from Ref. [37] with permission from the American Chemical Society. The data for DOPG/DOPC (1/9) are reprinted from Ref. [40] with permission from the American Institute of Physics.


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Figure 5: Leakage of AF647 and entry of CF-R9 into single DLPG/DTPC (2/8)-GUVs containing small vesicles, induced by CF-R9. (A) CLSM images of (1) AF647, (2) CF-R9, and (3) DIC for the interaction of 5.0 µM CF-R9 with the GUV. The numbers above each image show the time in seconds after the addition of CF-R9 was started. The bar corresponds to 10 µm. (B) Time course of the increase in the normalized fluorescence intensity of the GUV shown in (A). Red and green points correspond to the fluorescence intensity of the GUV lumen due to AF647 and that of the GUV rim due to CF-R9, respectively. (C) Dependence of Pentry (6 min) without pore formation on CF-R9 concentration. DLPG/DTPC (2/8)-GUV (○). For comparison, the data for DOPG/DOPC (2/8)-GUVs (●) (the same data in Figure 1C) are shown. Figure 6: Leakage of AF647 and entry of CF-R9 into single DLPG/DTPC (4/6)-GUVs not containing small vesicles, induced by CF-R9. (A) CLSM images of (1) AF647 and (2) CF-R9 for the interaction of 40 µM CF-R9 with the GUV. The numbers above each image show the time in seconds after the addition of CF-R9 was started. The bar corresponds to 10 µm. (B) Time course of the increase in the normalized fluorescence intensity of the GUV shown in (A). Red, green, and blue points correspond to the fluorescence intensity of the GUV lumen due to AF647, that of the GUV rim due to CF-R9, and that of the GUV lumen due to CF-R9, respectively. (C) Dependence of the fraction of leaked GUV at 6 min (□) and Pentry (6 min) without (or before) pore formation (○) on the CF-R9 concentration. The mean values and the standard errors are shown. Figure 7: Time course of the increase in rim intensity due to CF-R9, leakage of AF647, and entry of CF-R9 into single DOPG/DOPC/chol (2/6/4)-GUVs. (A) Interaction of 28 µM CF-R9 with DOPG/DOPC/chol (2/6/4)-GUVs containing small vesicles. CLSM images of (1) AF647, (2) CF-R9, and (3) DIC. The numbers above each image show the time in seconds after the addition of CF-R9 was started. The bar corresponds to 20 µm. (B) Time course of the increase in rim intensity of the DOPG/DOPC/chol (2/6/4)-GUV not containing small vesicles induced by 5.0 µM CF-R9, shown in Figure S4 (green □). The solid black line represents the best fit curve using eq. S3. The obtained value of kapp was 0.015 s-1. For comparison, time course of the rim intensity of the DOPG/DOPC (2/8)-GUV induced by 5.0 µM CF-R9 (green ∆) is shown. (C) Dependence of kapp on CF-R9 concentration. The mean values and standard errors of kapp are shown. The solid red line represents the best fit curve using eq. S4.


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Figure 8: Dependence of the free energy of a pre-pore, U (r), on the pre-pore radius for various line tensions, Γ: (A) 6.0 pN, (B) 12 pN, and (C) 18 pN. U (r) is calculated according to eq. 5 using σ = 3.0 mN/m.


Biochemistry

Figure 1 (A)

(C)

1.0

Fraction of entry

(B)

Fluorescence Intensity

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0.8 0.6 0.4 0.2 0.0 0

1.0 0.8 0.6 0.4 0.2 0.0 0

50 100 150 200 250 300 350 Time (s)

5

10 15 20 25 30 35 40 CF-R9 Conc. (µM)


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Figure 2 (A)

(B)

(C) 4

1.0 kapp ×10-2 (s-1)


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0.8 0.6 0.4

3 2 1

0.2 0.0

0

0

50 100 150 200 250 300 350 Time (s)

0

1

2 3 4 5 CF-R9 conc. (µM)

6


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Figure 3


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Figure 4 (A) 1.0

Pintact (t)

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100 200 300 400 500 600 Time (s)

(B)

0.01 -1

kp (s )

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0.001

0

1

2

3 4 5 σ (mN/m)

6

7

8


Biochemistry

Figure 5

(A)

(C)

(B) 1.0

Fraction of entry


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 38

0.8 0.6 0.4 0.2 0.0 0

50 100 150 200 250 300 350 Time (s)

1.0 0.8 0.6 0.4 0.2 0.0 0

5

10 15 20 25 CF-R9 Conc. (µM)


30

Page 35 of 38

Figure 6 (A)


(B) 1.0 0.8 0.6 0.4 0.2 0.0

0

(C) Fraction of entry and Fraction of Leaked GUV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

50 100 150 200 250 300 350 Time (s)

1.0 0.8 0.6 0.4 0.2 0.0 0

10

20 30 CF-R9 Conc. (µM)

40


Biochemistry

Figure 7 (A)

800

3.0

(C) kapp × 10−2 (s−1)

(B)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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600 400 200

2.5 2.0 1.5 1.0 0.5

0 0

0.0

50 100 150 200 250 300 350 Time (s)

0

2

4 6 8 10 CF-R9 conc. (µM)


12

Page 37 of 38

Figure 8

(C) Γ = 18.0 pΝ

40 30 U/kT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(Β) Γ = 12.0 pΝ

20 10

(Α) Γ = 6.0 pΝ

0 0

1

2

3 r (nm)

4

5


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 38

TOC graphics: Effects of lipid composition on the entry of cell-penetrating peptide oligoarginine into single vesicles Sabrina Sharmin, Md. Zahidul Islam, Mohammad Abu Sayem Karal, Sayed Ul Alam Shibly, Hideo Dohra, and Masahito Yamazaki


Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide

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